Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine. Affected individuals typically present with proximal tubulopathy (Fanconi syndrome) before one year of age and without specific treatment progress to end-stage renal failure by the end of the first decade. Cystine accumulation eventually leads to multi-organ dysfunction. The drug cysteamine reduces the intracellular concentration of cystine. However, the need for regularly spaced doses and a number of undesirable side effects render its administration difficult. Moreover, cysteamine does not prevent the proximal renal tubulopathy or the end- stage renal failure. The long-term objective of this project is to develop a new treatment for cystinosis by transplantation of autologous hematopoietic stem cells genetically modified ex vivo to express a functional CTNS gene. As pre- clinical studies, we will use the Ctns-/- murine model for cystinosis. These animals accumulate cystine and cystine crystals in all organs tested and develop ocular changes, neurological defects and kidney injuries similar to those observed in affected humans. Our preliminary data showed that transplantation of syngeneic whole bone marrow cells (BMC) or purified hematopoietic stem cells (HSC) expressing Ctns resulted in tissue engraftment of BMC or HSC-derived cells and significant reductions of cystine content in all the tissues tested. This treatment also prevented the development and progression of kidney dysfunction. We now propose to use Sca1+ HSC isolated from Ctns-/- mice and lentiviral vector (LV) for delivering the CTNS gene ex vivo. LV has proven its efficacy for long-term HSC transduction in mice but also in humans. In Specific aim 1, we propose to optimize the transduction of murine Sca1+ HSC using LV expressing reporter genes to enhance the safety and efficiency of a clinical trial as well as significantly reduce costs. In Specific aim 2, we will test if LV- transduced, CTNS-expressing HSC can prevent cystinosis-mediated tissue injury in young mice and potentially reverse cystinosis-mediated injury in older mice. The efficiency of these strategies will be tested by measuring CTNS expression and cystine content in different tissue compartments and by well-established functional studies to test the prevention or treatment of the kidney dysfunction, eye anomalies, bone anomalies and neurological defects. The immune response and safety of this strategy will be also tested as well as the toxicity of drug-mediated myeloablation in Ctns-/- mice. This work represents the first stem cell and gene therapy treatment strategies for cystinosis and builds the foundations for a future clinical trial. It also represents a proof of concept for autotologous HSC transplantation strategy to treat other lysosomal storage disorders. PUBLIC HEALTH RELEVANCE: Cystinosis is a hereditary disease characterized by the accumulation of cystine in all the cells of the body leading to cell death and tissue damage to kidneys, liver, eyes, muscle and brain. The long-term objective of this project is to develop a new treatment for cystinosis, the strategy is to use the patient's own bone marrow stem cells for transplantation and to genetically modify them ex vivo to introduce a functional version of the defective gene (CTNS). As pre-clinical studies, we will use the mouse model for cystinosis that accumulates cystine in all the tissues and develops similar defects to those of the human children.